The regulatory control of DNA replication and cell proliferation is one of the most fundamental processes in the life of an organism. Loss of cells's responsiveness to normal mechanisms of regulation results in cancerous growth of cells. The regulation of eukaryotic DNA replication is a complex process involving many protein-protein and protein-DNA interactions at various points. Among these are the commitment of cells to enter the S phase of the cell cycle, which is sensitive to a number of environmental influences such as the growth-stimulating factors and addition of serum. Such stimulation results in a pleiotypic response of events leading to DNA replication. One such response of cells is a marked increase in the level of the dinucleotide, diadenosine tetraphosphate, Ap4A. Ap4A is involved in multiple cellular events such as DNA replication and cell proliferation, DNA repair and platelet aggregation and vascular tonus. A protein that specifically binds to Ap4A has been detected in various prokaryotic and eukaryotic cells and we have found that in HeLa cells, this binding protein is part of a multiprotein DNA polymerase complex that is capable of replicating SV40 DNA in vitro. As part of our long-term goal of understanding the regulation of DNA replication in eukaryotes at the molecular level, we have proposed to study the physiological role of the Ap4A binding protein during DNA replication in this proposal. In the duration of this project, we will purify the binding protein to homogeneity, characterize the binding protein regarding its physical and biochemical properties, determine the role of this protein in SV40 in vitro DNA replication and the ability of Ap4A and the binding protein in initiating DNA replication. We will determine the amino acid composition and sequence of the binding protein and generate polyclonal and monoclonal antibodies to this protein. Utilizing these reagents, we will study the expression of this protein during various phases of the cell cycle and the intracellular location of this protein. Making use of the amino acid sequence data, we will generate oligonucleotide probes and clone the cDNA for the binding protein. In continuation this project, we will perform experiments to define the pbysiological role of this binding protein in the various cellular events that Ap4A modulates.